Abstract

Plant growth-promoting rhizobacteria (PGPR) are known to improve plant performance by

multiple mechanisms, such as the production of beneficial hormones, the enhancement of
plant nutritional status, and the reduction of the stress-related damage. The interaction
between plants and PGPR becomes of particular interest in environments that are
characterized by suboptimal growing conditions, e.g., high or low temperatures, drought, soil
salinity, and nutrient scarcity. The positive role of PGPR will become even more appealing in
the future, as world agriculture is facing issues as climate change and soil degradation. This
chapter aims to discuss the main mechanisms of the interaction between PGPR and plants and
will focus of how PGPR can decrease abiotic stress damage in cereals, which are critical
crops for human diet.

Keywords

PGR Bacteria, Global Warming, abiotic Stress. Legumes. Growth-Promoting Mechanism

 global warming
 abiotic stresses
 cereals1. Introduction

Global agriculture is facing the difficult challenge of increasing the productivity and output
required to feed a growing population. Additionally, fertile land areas available for
agriculture are gradually decreasing due to climate change, soil degradation, and pressure
from urban developments. These concerns are particularly relevant as they negatively affect
yields of cereal crops, which are a fundamental diet component in global society [1].

To help overcome this problem, researchers have turned their attention to understanding
interactions between plants and soil microorganisms. Plant roots interact with the soil
microbiota, which have various effects on plant growth and development, ranging from
beneficial to pathogenic [2]. Plant growth-promoting rhizobacteria (PGPR) play important,
but still poorly understood, roles in plant growth promotion, especially under environmental
stress such as drought, temperature, and salinity [2, 3, 4].
There are various mechanisms through which PGPR improve plant performance, often in a
synergic manner; some examples include the production of plant growth-promoting
hormones, improvement of plant nutritional status, and decreased stress damage [2].
Interactions between plants and PGPR can result in improvement of plant performance and
enhanced resistance to biotic and abiotic stresses which are important traits for cultivated
crops [5].

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2. Importance of cereals in global nutrition

Cereals are annual plants belonging to the monocotyledonous Poaceae family and are a vital
food source for humans as they provide almost one half of the calories that are consumed
daily in the world [6]. Furthermore, cereals are also extensively used as animal feed, mainly
for livestock and poultry, and as raw materials for many industrial processes, primarily the
production of alcoholic beverages [1].

In the last 50 years, the increase of cereal production (+240% in the time window 1961–2017
shown in Figure 1) is the result of increased yields per hectare (+201%) rather than the
expansion of land allocated to cereal production (+12%) (Figure 1). However, this trend has
recently decreased. The average production rate of cereals was 3.6% per year between 1961
and 2007, and it decreased to an average of 2.7% between 2007 and 2017 [7]. This is likely to
be linked to multiple factors, including climate change, soil degradation, use of soil for non-
alimentary purposes, restrictions on water, nutrients and land for agriculture, and limitations
of traditional breeding.

Figure 1.

Cereal cultivation records and world population data since 1961. Cereals cultivated land, soil
productivity as yield, world grain production, and world population are displayed [7].
3. Abiotic stress effects on agriculture

Most cultivated soils in the world are characterized as being suboptimal. Any deviation from
optimal growth conditions causes several interconnected reactions in plants that can be
described as an attempt to adapt to new environmental conditions in an effort to maintain
homeostasis. If the stress endures too long or is too severe, it can permanently damage plant
physiology or result in death. While many plants are able to adapt to stress, the process
requires energy that is diverted from active growth, resulting in smaller acclimated plants [8].
Abiotic stresses, that is, stresses caused by nonliving factors, are thought to be the main cause
of global crop loss with decreased productivity of more than 50% annually [9]. Drought and
salinity stress are potent environmental hazards for agriculture, particularly in arid and
semiarid regions which are already approaching the limits of crop productivity, and due to
global warming and degradation of agricultural soils, these regions may no longer support
crop plants in the future [10, 11].

3.1 Climate change

Food security is positively correlated with social and economic stability; given climate
change is threatening food production, there are extended and complex implications. Since
the mid-nineteenth century, average temperatures have increased by 0.8°C, and by the end of
this century, temperatures are predicted to increase between 1.8 and 4°C compared to the end
of the last century [12]. This change is causally related with human activities by the
production of greenhouse gases such as carbon dioxide, the concentration of which rose from
~284 ppm in 1832 to 397 ppm in 2013 [13].

While CO2 is generally accepted as a greenhouse gas, there is now increased interest in the
role of nitrous oxide (N2O). This compound can originate from the denitrification of N
fertilizers, which are commonly used in modern agriculture. In 2014–2015, more than half of
all N fertilizer was applied to cereal crops alone [14]. The reintroduction of N in N-depleted
soil is an essential agricultural practice that has led to increased yields over the last few
decades. However, the application of N fertilizer is inefficient, and it is estimated that only
one third of the applied N is absorbed by plants, with the excess being lost in surface runoff,
leaching in groundwater, or volatilization into the atmosphere [15]. Atmospheric N2O, while
less abundant than CO2, is 300 times more potent as a greenhouse gas [16].
Climate change caused by greenhouse gas emissions is predicted to directly impact the
productivity of agricultural systems in almost every part of the planet. While many
agricultural sites in cold-continental areas will benefit from the increased temperatures,
regions characterized by temperate, tropical arid, or subarid climates are likely to face
decreasing yields [17]. By modeling the effects of climate change on the yields of various
cereals in different areas of the world, it was predicted that by the end of the century, heat
stress events will increase in areas of Central and Eastern Asia, Southern Australia, Central
North America, and Southeast Brazil (rice); Northern India, the Sahel region, Southeast
Africa, and Central South America (maize); and Central Asia (wheat) [18]. Kompas et al.
[10] estimated that if no measures are taken to reduce greenhouse gas emissions, the average
world temperature increase of 4°C by 2100 will severely decrease food production in almost
all countries in the world. This will result in economic loss of approximately 23 thousand
billion US$ on average, with Southeast Asia and developing countries of Africa predicted to
face the largest losses (21 and 26% of GDP, respectively).

3.2 Agricultural soil degradation

Soil degradation is one of the main concerns impacting agricultural productivity, especially in
tropical and subtropical areas [19]. Globally, one third of land is affected by some form of
deterioration [20]. Unsuitable agricultural techniques, together with excessive crop residue
removal and unbalanced use of chemical fertilizers, can decrease soil quality, deplete organic
matter stocks, and increase erosion. Crop removal from the production site causes the loss of
elements that are essential for plant growth, and these elements must be constantly
reintroduced to avoid productivity decreases [21].

Using soils for agricultural purposes can cause degradation of water sources, due to leaching
of degraded fertilizers into groundwater. Many rivers in developing countries have severe
water pollution and eutrophication issues. Irrigation is an essential management strategy to
obtain sufficient productivity to meet food demands in many arid and semiarid areas, but it
can lead to undesirable effects. Improper irrigation techniques have increased saline-sodic
soils that now occur in more than 20% of irrigated lands [22].

4. Plant growth-promoting bacteria

A common misconception during the nineteenth century was that healthy plants should be
sterile, not interacting with any microorganisms. This assumption was initially questioned by
Victor Galippe [23], who proved that healthy plants could host various microbes in their
tissues. Today, we know that almost all terrestrial plants from various environments interact
with the surrounding microbiota during all stages of plant development. The relationship
between host plant and microbe can range from parasitism, commensalism or mutualism, or
neutral or beneficial for plant growth and can vary greatly due to a multitude of factors, both
biotic and abiotic. PGPR are attracted to plants by organic exudates released through roots
and colonize the root surface and the soil directly in contact with the root. The soil matrix
directly in contact with plant roots is called the rhizosphere [24], and the extracellular surface
of roots is termed the rhizoplane [25]. Here, colonizing microorganisms can establish the
exchange of nutrients and various compounds with the plant, summarized in Figure 2.

A model of interactions between plants and PGPR. Exudates released by plant roots attract
soil bacteria that can colonize rhizosphere and/or plant tissues. Here, they provide various
beneficial compounds to the plant in exchange of nutrients, mainly photosynthates.

Nutrients and organic compounds released into the rhizosphere from roots are derived from
photosynthesis, and plants release up to 30% of their photosynthates through the roots [26].
These include a variety of compound classes such as carbohydrates, amino acids, organic
acids, flavonoids, and lipids that can be used as energy sources for microbes [27]. The
sensing and active migration of bulk soil bacteria toward these compounds is called
chemotaxis, leading bacteria to colonize the rhizosphere and rhizoplane [28]. By producing
exudates, plants can select bacterial species that are attracted to specific compounds, thereby
directing the abundance and diversity of microbes in the rhizosphere [29, 30]. Wild oat has
been reported to modify the bacterial population of its rhizosphere enriching mainly the
Firmicutes, Actinobacteria, and Proteobacteria [31]. The latter group in particular is
commonly believed to be the main microbial component in PGPR interactions, due to their
capacity for fast growth and diverse metabolic pathways capable of utilizing a great variety of
exudate compounds as an energy source [29]. In the model cereal plant Brachypodium, the
rhizosphere microbiome changes not only within the loosely bound rhizosphere soil and
tightly bound rhizosphere soil but also within seminal and nodal roots [32]. It is noteworthy
that plants can indirectly influence the colonization of the rhizosphere, by changing the
environment conditions. Some examples are changes in pH levels by ion uptake, the
reduction of O2 and H2O levels caused by root respiration, and water absorption [29].

Different types of root exudates can attract different PGPR. For example, various strains
of Azospirillum brasilense, a gram-negative Alphaproteobacteria, showed different degree of
attractions to various compounds released by different host plants [33]. The composition of
root exudates can vary greatly among different plant species. Two different studies [34, 35]
reported how even different genotypes of the same plant species can host different bacterial
populations in their rhizosphere. Exudates vary between different parts of the roots, different
developmental stages of the plant, or as a response to different growth conditions [36]. This
means that the same plant can interact with a multitude of different soil bacterial strains over
time and space [37].

Nehl et al [38] use the term “rhizobacteria” to describe rhizoplane/rhizosphere bacteria, but
there are also endophytic bacteria that can reside inside plant tissues. To date, numerous
interactions between plants and rhizosphere-/rhizoplane-colonizing bacteria have been
described, but some microbes are even more specialized. Once they have colonized the
rhizoplane, they are able to penetrate root tissues and directly access apoplastic organic
compounds, thereby avoiding competition with other microbes in the rhizosphere [39]. Root
penetration can be both active, by the production of cell wall-degrading enzymes such as
cellulase, and passive, for example, entering via the cracks that form on the root surface
during lateral root development [40]. Colonization beyond the rhizosphere into the apoplast
requires specialized microbial morphology. Czaban et al. [41] described how the occurrence
of flagellar motility in bacterial strains isolated from the internal root tissue of wheat was five
times higher than what was observed in bacteria isolated from the rhizosphere.

Bacillus, Pseudomonas, Enterobacter, Klebsiella, Serratia, and Streptomyces are some of the

most commonly found genera of endophytic bacteria in plant tissues [42]. By passing the
endodermis, many bacterial species are able to spread from the roots, reaching and colonizing
other organs of the stems [43]. Endophytic bacteria can also spread from plant tissue to seeds
becoming the starting inoculum for the colonization of subsequent generations of plants. The
transmission of bacteria through generations of plants is a process known as vertical
transmission. Johnston-Monje and Raizada [44] described how modern varieties of maize and
their wild ancestors share common endophytic bacteria communities hosted in their seeds,
and a following study conducted on wheat demonstrated how these communities play a
positive role in plant growth [45].

4.1 Plant growth promotion driven by rhizobacteria

Galippe’s intuition that plants interact with microbes throughout their life led to a significant
increase in the comprehension of the beneficial role that bacteria can have on plant growth.
PGPR interactions can result in higher plant biomass, higher nutritional value, better survival
rates, and generally require lower agricultural inputs. Focusing on cereals, PGPR can
significantly improve plant performance in several environments, particularly those
characterized by suboptimal growth conditions. Some of the main benefits that plants obtain
are increased root development which imparts improved resistance to temperature and
osmotic stress, soil pollutants, pests, and pathogens [46].

It is well established that plant responses to biotic and abiotic stresses require complex
adaptations to structure and metabolism. When biotic and abiotic stresses are applied
simultaneously, plants respond much differently compared to stresses applied separately [9].
It is therefore reasonable to assume if a plant is exposed to both biotic and abiotic stresses
that PGPR may directly mitigate the effect of biotic stresses by improving plant resistance to
abiotic stresses.

4.1.1 Hormone-related mechanisms

The most well-described mechanism by which PGPR can improve cereal productivity is the
productions of various plant growth-promoting hormones that usually co-affect the
performance of the plant in a highly integrated manner [47]. Auxins are a class of hormones
typically synthesized by apical buds, and from there they are transported to other parts of the
plant. In this class of hormones, the most characterized is indole-3-acetic acid (IAA), which
enhances cell elongation and differentiation and, in roots, stimulates lateral root development
[42, 48]. Various reports have shown how the production of auxins from PGPR is one of the
most important mechanisms for plant growth promotion. Barbieri and Galli [49] inoculated
wheat with two strains of Azospirillum brasilense, of which one was a mutant with impaired
IAA production. They observed how only the wild-type strain promoted lateral root
development, a result that suggests a primary role of IAA in improving plant root
development. IAA can indirectly improve the nutritional status of the plant by increasing root
development (specifically lateral roots), hence allowing the plant to explore a higher portion
of soil substrate, an important trait particular for the acquisition of low-mobility nutrients
such as phosphorus [50].

Gibberellins (GAs) can be produced by PGPR [51] and are believed to play an important role
in promoting plant growth. These diterpene hormones are naturally present in plants,
regulating key processes such as seed germination, stem elongation, leaf expansion, root
growth, and root hair abundance [52, 53]. One of the best known GAs is GA 3, commonly
known as gibberellic acid, which plays a key role in determining plant source-sink relations.
The role of gibberellins in the response of cereals to stresses varies depending on the stress
type [54], but in general, plants tend to reduce GAs levels when growing in suboptimal
conditions. The exogenous application of gibberellins has been reported to improve wheat
and rice performance undergoing saline stress [55, 56] and to reduce heavy metal stress
symptoms in rice [57].

Many PGPR are able to degrade 1-aminocyclopropane-1-carboxylic acid (ACC) through the
enzyme ACC deaminase and use the degradation products as a nitrogen source [42]. ACC is
the biosynthetic precursor of ethylene, a hormone naturally present in plants, and its
abundance is often increased in response to stresses. While at optimal levels, ethylene is
involved in essential processes such as tissue differentiation, root development, flowering,
grain development, and natural tissue senescence and abscission; when overproduced it can
decrease plant performance [58]. In abiotically stressed plants, the increase of ethylene can
trigger chlorosis and early maturation and senescence of organs, seeds in particular [59, 60],
and have an inhibitory effect on root growth [42]. By impairing the ethylene signaling
pathway, the interaction with PGPR can decrease the stress-related damage in the plant [2].

Similar to ethylene, abscisic acid (ABA) is a hormone commonly produced by plants in

response to various types of stress, particularly osmotic stress [61]. Naturally involved in
seeds and buds dormancy, ABA shares the first biosynthetic steps with cytokinins, a
phytohormone class that often plays an antagonistic role to ABA. In dry or saline soils,
reactive oxygen species (ROS) increase the biosynthesis of ABA, which is then transported to
leaves, where it causes stomatal closure to reduce transpiration and water loss [62]. As a
consequence, the diffusion of CO2 into leaves is decreased, lowering photosynthetic rates
[63, 64]. PGPR have been reported to increase the resistance of plants to salinity, hence
decreasing the stress-related ABA accumulation in plants and preserving photosynthetic
efficiency [65, 66].

5. Plant-bacterial interactions enhance abiotic stress responses

Bacteria can have various effects on their host plant. PGPR can affect plant growth both
directly, such as by fixing atmospheric N 2 into biologically available N compounds or by
producing growth-promoting hormones [52], and indirectly, by preventing the growth of
plant pathogens or increasing plant resistance to them [43]. A necessary condition for bacteria
to be beneficial to a plant is rhizosphere competence as the competition and conditions in the
rhizosphere are vastly different to that of bulk soil. The rhizosphere contains a higher
abundance of bacteria than bulk soil, but the diversity is much lower. The colonization of the
root system of plants is not homogenous; the density of specific bacteria varies in different
parts of the root system and is likely to be related to different root exudates released by
different parts of the roots [37]. Another mechanism likely to regulate the colonization of the
rhizosphere is bacterial quorum sensing, which is the regulation of gene expression driven by
bacterial population density and can occur both within bacteria of the same species and
among different species [67]. Quorum sensing can influence the bacterial competitiveness,
therefore affecting the roots colonization patterns [37].

5.1 Thermic stress adaptation

Temperature stress causes a shift in hormone production, particularly ethylene, which can
often impair plant growth [58]. High-temperature stress causes denaturation and aggregation
of cellular proteins that, if left unchecked, leads to cell necrosis. Imbalance between ABA
and cytokinins derived from prolonged heat stress during the reproductive stage can lead to
grain abortion [68]. Heat responses include inhibition of normal transcription and translation
and increased expression of genes coding for heat shock proteins and thermotolerance
induction [69]. Low-temperature stress, conversely, damages metabolic processes, changes
membrane properties, causes structural changes in proteins, and inhibits enzymatic reactions
[70]. If it occurs during spore formation, cold can cause sterility of flowers by interfering
with meiosis [71].

The literature on PGPR interactions with cereals at suboptimal temperatures is relatively

scarce, and the mechanisms by which cereals adapt are not well defined. It is suggested that
the geographical origin of the bacteria determines the optimal growth range at which they
interact beneficially with plants. In a study on wheat, bacteria isolated from cold climates
have been reported to efficiently colonize the plant rhizosphere and improve their resistance
to low-temperature stress, and the same trend was observed when wheat plants were
inoculated with bacteria isolated from warm environments and subjected to high-temperature
stress [72]. It is possible that the bacteria isolated from different temperatures can outcompete
the indigenous microbial population by tolerating either cold or warm conditions giving rise
to a higher abundance and colonization of the rhizosphere.

Inoculation with a Pseudomonas aeruginosa strain isolated from a hot semiarid environment

improved survival rate, development, and biochemical parameters of sorghum seedlings
when the plants were exposed to heat treatment, while the biomass production was not
affected at optimal temperatures [73]. In another study, various cold-
tolerating Pseudomonas spp. were inoculated onto wheat grown at low temperatures, giving
analogous results. The authors suggest the beneficial effect was linked to a better root
development in inoculated strains that improved nutrient uptake and, in general, caused a
better adaptation to cold [74].

As global warming threatens to change significantly the temperature of most cultivated lands
[17], the development of cereals with enhanced adaptation capacity to heat or cold stress is an
essential task in order to sustain profitability and production at suboptimal temperature
conditions. While further research is necessary to better understand the mechanisms that
regulate PGPR-plant interactions in such conditions, the studies done so far suggest how
PGPR can be a valuable source of temperature-stress resistance, especially when they
evolved in areas characterized by warm or cold climates, depending on the case.

5.2 Osmotic stress adaptation

Both dry and saline soils can cause osmotic stress in plants, which results in cell dehydration
due to lack of water (drought) or unavailability of water (salinity). These two stresses are
often agronomically significant, as high salinity in soil is mainly caused by irrigation, a
necessary practice for increasing yields in many areas of the world characterized by
insufficient rainfalls. When water supply is insufficient to remove ions from superficial soil
layer, they accumulate causing an increase of salinity [75].
Salinity is also the result of land clearing, as deep subsurface roots no longer are able to keep
the water table below ground level. As the water table rises, it brings with it saline water that
can render hundreds of square kilometers of agricultural land uncultivable [76]. Plants
growing on such soils often suffer from osmotic stress that reduces water absorption and
increases ionic concentration in tissues to toxic levels [77]. PGPR can decrease these stress
symptoms through various mechanisms, such as production of Na+-binding
exopolysaccharides [78], improvement of ion homeostasis [79], decrease of ethylene levels in
plants through ACC deaminase [80], and synthesis of IAA [81]. Wheat seeds inoculated with
a species from the genus Pseudomonas showed increased germination rates in a saline
environment; Egamberdiyeva [82] ascribed this to the production of plant growth regulators
by the bacteria.

Drought is considered as the major cause of yield loss [83], negatively affecting most
physiological processes in plants. Plant cells respond to water loss by increasing the
production of abscisic acid (ABA) in roots that increases water uptake and causes leaf
stomatal closure and reduces leaf expansion to reduce dehydration [84]. Smaller leaves cause
impaired photosynthesis, consequently decreasing dry matter accumulation and grain yield
[85]. Under water deficiency, both cell division and enlargement are lowered due to damaged
enzyme activities, leading to overall smaller plant organs. Grain production is also reduced in
cereals due to flower abortion [86, 87].

Plants often react to drought by increasing the amount of osmolytes in their tissues and
consequently increase their osmotic potential [88]. Drought can also cause an increase of
ROS in plant tissues. Proline, an amino acid whose abundance is increased under water
deficiency, can both work as an osmolyte and scavenger for ROS under stress [89]. In
general, PGPR can improve the performance of plants in dry environments by exudating
osmolytes that increase the osmotic potential of plants [42, 90, 91].

Another mechanism for improving resistance to drought is the synthesis of beneficial

hormones (IAA) and enzymes (ACC deaminases) and the decrease of stress-related hormones
such as ethylene and ABA in the plant. Naveed et al. [92] reported that two maize cultivars
exposed to drought showed reduced damage when inoculated with two different PGPR,
probably due to hormones produced by the bacteria and stress-reducing enzymes synthesized
by both the plants and the bacteria during the interaction. Wheat plants inoculated with
various PGPR showed an improved resistance to salt and drought treatments, linked to
decreased ABA and ACC levels in plant tissues [65]. In a similar study [66], rice plants
showed decreased endogenous ABA levels and increased biomass when inoculated
with Bacillus amyloliquefaciens; the authors hypothesize that inoculation increased salt
tolerance in plants through an ABA-independent pathway, and this prevented the stress-
dependent ABA accumulation and the resulting growth impairment [63].

Sarig et al. [93] report that sorghum plants subjected to osmotic stress after their emergence
showed decreased damage when colonized by Azospirillum brasilense. It is unclear, however,
if the observations were a drought-specific response or an indirect effect of inoculated plants
showing a better root development and higher hydraulic conductivity at the time of the stress.
In two successive studies [94, 95] conducted on various Azospirillum spp., inoculated wheat
plants subjected to drought had decreased grain loss, better water status, and higher K and Ca
content, with the latter in particular suggested to be involved in the adaptation of the plants to
environmental stress. Bacterial nitrate reductase was also suggested to play an important role
in nitrate assimilation of plants under drought [95].

As previously mentioned, drought and saline stress are related, since salinity is often the
result of irrigation practices to avoid plant desiccation from drought stress. This concern may
become more relevant in future years, as higher temperatures caused by global warming will
result in higher evapotranspiration, hence requiring increased irrigation. By the year 2050,
50% of all arable lands might be affected by serious salinization [96]. Improving the
resistance of plants to dry environments would decrease the necessity of irrigation, indirectly
decreasing the ongoing salinization process in agricultural land.

5.3 Improvement of the plant nutritional status

In natural environments, plants die and decompose where they grew, and the subsequent
detritus reintroduces soils with most of the nutrients they absorbed during their growth. In
cultivated lands, those nutrients are removed at harvest and must be constantly replaced to
avoid productivity decrease. Among the macronutrients, nitrogen, phosphorus, and potassium
are the most important for plant growth, and they are typically reintroduced using synthetic
fertilizers. Unbalanced use of fertilizers can decrease soil quality, consume organic matter
stocks, and increase erosion risk. Soil bacteria can improve the nutritional status of plants
directly by increasing nutrient bioavailability and/or indirectly by improving plant root
development, hence allowing them to explore higher areas of soil [97].
5.3.1 N2 fixation and absorption

Several bacterial species are classified as diazotrophs, which are microorganisms that are able
to utilize the nitrogenase enzyme to fix atmospheric N 2. Diazotrophic bacteria can fix N2 in
either a free-living form or in association with a host as an endosymbiont. The most well-
described interaction between plants and diazotrophic bacteria is the rhizobia-legume
symbiosis. Rhizobia are a group of various Proteobacteria that can colonize plant roots and
fix atmospheric nitrogen, which is then partly provided to the plant in exchange of
photosynthates [98]. While this association has been observed mainly in legumes, some
species of rhizobia can also colonize cereals. Gutierrez-Zamora and Martinez-Romero [99]
showed how maize and bean plants cultivated in association shared the same Rhizobium
etli strains, with the bean plants probably constituting the source of inoculum for maize. The
interaction with the rhizobia increased the biomass of both crops, but in maize this outcome
might have been linked to mechanisms other than N 2 fixation, such as hormone production.
Rice inoculated with an Azoarcus sp. showed improved growth regardless of colonization by
the wild-type strain or with a mutant strain deficient in the nitrogenase genes [3]. When
spring wheat and maize were inoculated with two different rhizobia and grown at various soil
N levels, the two strains were effective in enhancing plant growth only at low and
intermediate levels of soil N. The authors suggest that plant growth-promoting hormones
released by the bacteria caused a better root development in inoculated plants that were able
to absorb more nutrients from the soil [100].

In general, diazotrophic bacteria associated with cereal roots often carry the nitrogenase
genes necessary for the fixation of atmospheric nitrogen, but the relative enzymes are not
always synthesized inside plant tissues. Furthermore, the amount of fixed N provided to the
plant is often negligible, due to low presence of diazotroph bacteria or because bacteria use
fixed nitrogen for their own growth [101]. The nitrogenase enzyme cannot function in the
presence of O2, so it may be desirable to engineer free-living diazotrophic bacteria that are
able to colonize plant tissues. Other possible ways might be to increase the fixing bacteria
population by engineering plants capable of exudating diazotroph favorable compounds or
engineering bacteria capable of providing the plant with higher levels of nitrogen [102].

Fox et al. [103] modified a Pseudomonas sp. genome by adding a gene cluster with
nitrogenase activity that improved the performance of wheat and maize by fixing N 2. This is
an example of some of the approaches toward nitrogen-fixing cereals, that is, plants capable
of sourcing the N necessary for their growth from the atmosphere via endosymbionts [104].

Farmers have benefited from the rhizobia-legume symbiosis for centuries, and extending this
characteristic to cereals would be a decisive benefit for modern agriculture, providing a
continuous, ecologically, and economically sustainable source of N to the most important
crops.

5.3.2 Improvement of soil nutrient uptake

Despite the benefits PGPR impart on plant nutrient content, it is often unclear if this
improvement is related to an enhanced mineral uptake or if it is the result of improved root
system development in inoculated plants due to bacterial hormones and/or enzymes [48].

Various bacterial strains are known to increase bioavailability of phosphorus in soil, due to
the mineralization of organic phosphate and solubilization of inorganic phosphate. Some of
the bacterial compounds linked to these two processes are acid phosphatases and organic
acids, respectively [105]. Phosphate-solubilizing bacteria have been reported to improve the
growth of maize [106], rice [107], and wheat [108].

PGPR can also synthesize siderophores that are low-molecular-weight compounds with high
iron-binding affinity [109] that can complex with Fe (predominantly Fe3+) in soil. The iron-
siderophore complex is then assimilated by the bacterium using a complex-specific receptor
[110]. This has various effects, it depletes the soil iron supply, thereby preventing the growth
of other potentially pathogenic microbes, and, if the iron is then provided to the plant, it can
directly improve plant growth [48]. Furthermore, the bacterial nitrogenase activity
and nif gene expression are iron dependent [111, 112]; hence, the absorption of iron from the
soil enables diazotroph bacteria to convert atmospheric N2 to a form that is bioavailable for
the plant.

PGPR can indirectly improve plant performance neutralizing the stress-related hormones
produced by the plant in poor soils. Wheat plants grown at various levels of N, P, and K,
showed increased grain yield and biomass production when colonized by Pseudomonas spp.,
with the bacterial growth promotion being negatively correlated with the amount of provided
nutrients [97]. The authors ascribe this outcome to bacterial production of ACC deaminase
that decreased ethylene levels produced by plants as a response to low nutrients levels, which
impaired root development in uninoculated plants.

Overall, plant growth promotion is ascribed to a combination of multiple mechanisms.

Egamberdiyeva [113] inoculated maize seeds with PGPR with nitrogenase and/or IAA
activity and grew them on two soil types with different nutrient availabilities. Inoculated
plants generally developed a higher root and shoot biomass and had higher N, P, and K
contents, the improvement being more pronounced in plants grown on nutrient-poor soils.
However, this study did not consider the possible interactions of inoculated strains with the
native microbial populations that may have affected the results.

In 2014–2015, out of 182 million metric tons (Mt) of consumed fertilizer, one half was
applied to cereals [14]. Cereals consumed more than one half of N fertilizers and more than
one third of P and K fertilizers. As previously mentioned, these amendments have both a high
economical and environmental cost, as they can cause soil degradation, pollution of water,
and eutrophication. While developing N-fixing PGPR is a task yet to be achieved in cereal
agriculture, it is well documented how PGPR can improve the efficiency of nutrient uptake in
crops. This can occur by either increasing the bioavailability of nutrients in the soil or as a
consequence of better root development, resulting in better soil exploration.

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6. Issues and perspectives

Cereal-PGPR interactions have been widely studied over the last few decades, and the
positive influence that they can have on plant growth is still being established. However, the
lack of consistency among different studies is still a concern, highlighting that when multiple
biological actors are involved, no generalizations can be made. The same bacterial strain can
be beneficial to a plant species and damage another [114] or have no effects or even be
detrimental for plant performance when the growing conditions are optimal but become
beneficial when growing conditions worsen [2, 3, 4]. In two studies on maize and rice
subjected to water deficiency [90, 115], the beneficial effects of various bacterial isolates on
plant growth increased with the severity of the stress. Studying the interaction between PGPR
and gum rockrose (Cistus ladanifer), Solano et al. [116] hypothesized that a possible
explanation for this is that poor environments may impair the growth of indigenous microbial
communities, this way decreasing the competition for those microbes that establish
advantageous relationships with plants. Another possible explanation is that when the main
bacterial mechanism of plant growth promotion is providing them with nutrients, the benefit
might be limited in nutrient-rich soil, while it can be significant in the case of limiting
nutrients [117].

The observed outcomes change particularly from laboratory and climate chamber trials to
more open setups such as greenhouses and field, in which bacteria often fail to improve plant
growth [37]. Most of the studies conducted so far on the interaction between cereals and
PGPR were performed in controlled environments, usually applying only one single stress at
a time. While this is a necessary compromise when starting to study this interplay, it often
entails a significant bias from realistic field environment [2], in which plants frequently face
more variable growing conditions and face multiple stresses at the same time, triggering
unique responses in plants that are different from the sum of plant response to stress applied
individually [118]. So far, very few experiments have studied the interaction between bacteria
and crops under multiple stresses, but replicating as accurately as possible real field
conditions is an essential step for understanding and exploiting the role of PGPR in
agriculture. In addition to the more unstable growing environment, another important variable
added in field experiments is the interaction with the native microbiota. Often inoculated
bacteria in the field show lower rhizosphere or root colonization than laboratory, climate
chamber, and greenhouse trials [119], in which the growth medium is usually sterilized at the
beginning of the experiment.

One of the hypotheses that can be drawn from the current literature is that the origin of the
inoculated bacteria is often a decisive factor for the interaction to improve plant growth.
Bacteria isolated from the same plant species used in trials are more likely to play a beneficial
role, probably due to the plant-specific exudates that have a key role in the early phases of the
interaction [100]. Similarly, bacteria isolated from environments characterized as suboptimal
(temperature in particular) that are similar to the conditions and stress applied in plant trials
may be more beneficial than bacteria isolated from optimal conditions, delivering more
benefits to the plant, due to adaptations that allow the bacteria to be more competitive than
the native microbiota [72]. Unfortunately, inoculum used in trials may become less effective
due to continual cultivation in laboratory environments, and when planning a plant trial, this
should be taken into consideration.
One of the problems facing commercialization of PGPR on markets is the inoculation
delivery method on plants. In the laboratory, a common method is dip inoculation where
seedling roots are immersed in bacterial culture and then transplanted into the growth
substrate, but this approach is not feasible for annual cereals on the field scale. The on-field
application of bacterial solutions after seedling germination, while less laborious, still
requires considerable equipment and technical knowledge. The most feasible way to apply
PGPR on field is probably the use of pre-inoculated seeds (this is already used for rhizobia-
legume inoculation) allowing farmers to bulk sow, relieving them from the inoculation step.
When the seed bacterial treatment is done immediately before germination, the required
strength of bacterial inoculum is typically smaller than in seedling treatments, but ideally
inoculants should survive long enough on seed coats to be present during germination;
however, prolonged survival of microbial treatment on seeds is still a challenge [120].
Moreover, inconsistencies between performances of seed inoculants are often observed in
different trials, and further research is required to address this issue [121]. Utilizing vertical
transfer of microbial endosymbionts in seeds may also present a possible inoculation
technology that has not been explored extensively and may provide economic benefits to
farmers [120] and could potentially mitigate the problem of inoculum viability in seed coats.
Recently, studies on bacterial strains vertically transmitted in cereal seeds have shown
promising plant growth-promoting effects, likely linked to their ability to solubilize
phosphorus, produce hormones, siderophores, and ACC deaminase [122]. By exploiting the
existing interactions between plants and known seed endophytic bacteria or isolating new
bacterial strains capable of inhabiting seeds for vertical transmission by crops, new
technologies may emerge that have large-scale economical applications.

During the last decades, selection of crops has been driven by increased productivity in
nutrient-rich environments, with scarce focus on the positive effects of PGPR, and this trend
might have led to the loss of plant traits associated with the microbial interaction [5]. The
identification and reintroduction of the genes associated with those traits might enhance the
positive effects of PGPR, especially in poor environments, and selecting plants that have
superior interaction with rhizosphere microbiota should be considered in plant breeding
programs. Additionally, a more immediate way to alleviate temperature stress could be to
inoculate plants with bacteria originated from hot-climate regions that as a consequence are
more likely to help their host to perform better in a warming environment [29, 72].
The interaction with microbes will gain more attention in the future, considering the effects
of climate change, due to the microbial genetic plasticity compared to plants. PGPR may
evolve rapidly, developing efficient adaptation strategies to the benefit of the plant host as
well.